associations between brain microstructures metabolites and cognitive deficits during chronic hiv 1 infection of humanized mice

Longitudinal open field activity tests demonstrated lack of habituation indicating potential for memory loss and persistent anxiety in HIV-1 infected mice compared to uninfected controls

R E S E A R C H A R T I C L E Open Access

Associations between brain microstructures,

metabolites, and cognitive deficits during chronic HIV-1 infection of humanized mice

Michael D Boska1†, Prasanta K Dash2†, Jaclyn Knibbe2, Adrian A Epstein1,2, Sidra P Akhter2, Natasha Fields2,

Robin High3, Edward Makarov2, Stephen Bonasera4, Harris A Gelbard5, Larisa Y Poluektova2,

Howard E Gendelman2,4*and Santhi Gorantla2

Abstract

Background: Host-species specificity of the human immunodeficiency virus (HIV) limits pathobiologic, diagnostic and therapeutic research investigations to humans and non-human primates The emergence of humanized mice as a model for viral infection of the nervous system has overcome such restrictions enabling research for HIV-associated end organ disease including behavioral, cognitive and neuropathologic deficits reflective of neuroAIDS Chronic HIV-1 infection of NOD/scid-IL-2Rgc

null

mice transplanted with human CD34+hematopoietic stem cells (CD34-NSG) leads to persistent viremia, profound CD4+T lymphocyte loss and infection of human monocyte-macrophages in the meninges and

perivascular spaces Murine cells are not infected with virus

Methods: Changes in mouse behavior were measured, starting at 8 weeks after viral infection These were recorded coordinate with magnetic resonance spectroscopy metabolites including N-acetylaspartate (NAA), creatine and choline Diffusion tensor magnetic resonance imaging (DTI) was recorded against multispectral immunohistochemical staining for neuronal markers that included microtubule associated protein-2 (MAP2), neurofilament (NF) and synaptophysin (SYN); for astrocyte glial fibrillary acidic protein (GFAP); and for microglial ionized calcium binding adaptor molecule 1 (Iba-1) Oligodendrocyte numbers and integrity were measured for myelin associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG) antigens

Results: Behavioral abnormalities were readily observed in HIV-1 infected mice Longitudinal open field activity tests demonstrated lack of habituation indicating potential for memory loss and persistent anxiety in HIV-1 infected mice compared to uninfected controls End-point NAA and creatine in the cerebral cortex increased with decreased MAG NAA and glutamate decreased with decreased SYN and MAG Robust inflammation reflected GFAP and Iba-1 staining intensities DTI metrics were coordinate with deregulation of NF, Iba-1, MOG and MAG levels in the whisker barrel and MAP2, NF, MAG, MOG and SYN in the corpus callosum

Conclusions: The findings are consistent with some of the clinical, biochemical and pathobiologic features of human HIV-1 nervous system infections This model will prove useful towards investigating the mechanisms of HIV-1 induced neuropathology and in developing novel biomarkers and therapeutic strategies for disease

Keywords:1H magnetic resonance spectroscopy, Behavioral and cognitive deficits, Diffusion tensor imaging, HIV-1, Humanized mice

* Correspondence: hegendel@unmc.edu

†Equal contributors

2

Department of Pharmacology and Experimental Neuroscience, University of

Nebraska Medical Center, Omaha, NE 68198, USA

4

Department of Internal Medicine, University of Nebraska Medical Center,

Omaha, NE 68198, USA

Full list of author information is available at the end of the article

© 2014 Boska et al.; licensee BioMed Central This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,

Persistent HIV-1 infection commonly leads to cognitive,

behavioral and motor abnormalities called HIV-associated

neurocognitive disorders (HAND) [1-3] Despite intensive

research, investigations seeking virus-associated central

nervous system (CNS) biomarkers and HAND therapies

have failed, in measure, due to the multifactorial nature of

disease and by few relevant small animal models [4-6]

The obstacles in generating a small animal model require

that viral tropism, neuroimmune activation, cognitive

im-pairments and CD4+ T cell losses are operative [1,7,8]

This has remained an unmet goal Added to these

obsta-cles in mirroring human disease are the concomitant use

of abuse drugs, common opportunistic infections, hepatic

dysfunction, nutritional deficiencies, social demographics,

ongoing antiretroviral therapies, psychiatric illness, aging

and altered immune responses often seen in infected

humans [9] The shift in human disease severity, from

overt dementia to subtle cognitive dysfunction, affects

changes disease demographics and as such has also made

modeling of human disease even more complex [10,11]

This has occurred as a consequence of the wide spread

use of antiretroviral therapy (ART) [3,12]

Despite considerable improvements in disease severity

up to half of infected patients show deficits in memory

and psychomotor functions Disease can readily be seen

through neuropsychological testing using common

met-rics of cognitive function [13-16] Such deficits are

im-portant as they can deeply affect the quality of life and

making research into ways to find better diagnostics and

therapeutic interventions timely and important [17-19]

However, in order to accomplish these goals model

sys-tems of current human disease are needed Divergent

viral-induced immune and cognitive deficits seen as a

consequence of viral infection and ART need be

consid-ered Indeed, early evidence that the terminal stages of

end organ HIV disease could be reflected in rodent

models of neuroAIDS was realized, over the past decade,

within our own laboratories [5,20-23] However, despite

creating such models, no cross validation of the

intersec-tion between immune and behavior abnormalities were

realized If this were possible, such a model could speed

the discovery of new pathways of disease towards better

understanding viral pathogenesis and even allow early

diagnostic studies [5,24,25] To these ends, we now

dem-onstrate, for the first time, cross species transference of

immune and viral factors that lead to neurocognitive

deficits, neuropathology, brain metabolite alterations,

and brain subregion damage in virus-infected

NOD/scid-IL-2Rgc null humanized mice (CD34-NSG) As such the

work represents a substantive step forward from current

models [22] Remarkably, the observed metabolic [proton

magnetic resonance spectroscopy (1H MRS)],

microstruc-tural [diffusion tensor magnetic resonance imaging (DTI)],

histologic and behavioral aberrations that characterize hu-man disease are partially replicated in the mice and as such mirror components of neuroAIDS The model none-theless provides unique insights in the biomolecular mech-anisms of how HIV-1 infection affects neural function

Results

Overview

Humanized mice for studies were divided into replicate HIV-1 infected and uninfected animal groups Imaging was performed and blood samples acquired from all ani-mals prior to and from four weeks to 16 weeks after in-fection At 16 week animals were perfused with normal saline and the brains were harvested for histopatho-logical evaluations The experimental scheme is outlined

in Figure 1 The overall experimental design enabled cross validation of all bioimaging, viral and, immune tests with brain histopathology for each animal In paral-lel experiments, mice were evaluated for behavior by open field activity (OFA) These were chosen to reflect memory and anxiety in mice Using replicate animals for behavioral tests avoided confounding influences of the anesthesia which was required for all bioimaging tests

Immunologic and virologic features of HIV-1 infection in humanized mice

CD34-NSG mice were monitored prospectively for blood CD4+and CD8+ T cells (Figure 2A) At 22 weeks of age, mice (n = 20) were infected with HIV-1ADA VL and CD4+ and CD8+ T cells were analyzed at 4, 8, 12 and 16 weeks after infection (Figure 2B) from the 100 μl of blood col-lected immediately following MRS and DTI studies Steady decreases in total CD4+T cells with concomitant increases

in CD8+cells were observed (Figure 2A) during the course

of HIV infection Viral load (VL, copies/ml blood) peaked

at eight weeks after viral infection and all infected animals had sustained VL (7.05 × 105to 1.5 × 107) until 16 weeks where reductions in total human cells (as measured by CD45) mirrored decreases in VL (4.77 × 103to 5.1 × 106) Replicate uninfected controls (n = 10) demonstrated stable CD45 and CD4/CD8 ratios throughout the study

VL and identifications of infected cells

To assess the total number of human cells and VL in the brain (copies/cc tissue), we dissected the cortex, ex-tracted RNA, and real time RT-PCR performed for hu-man CD45 and HIV-1gag Comparison of VL in brains (Figure 2C) with their peripheral blood VL at 16 weeks (Figure 2D) showed that 3 animals with low peripheral

VL have significant levels of HIV-1gag in brain, indicat-ing that peripheral viral loads are not correlated to brain levels (r = 0.40, p = 0.264, Spearman correlation) The data is expressed as total HIV-1 RNA copies/μg total RNA after normalization with GAPDH We identified

the cell targets for viral infection in the periphery (spleen)

and nervous system (brain) In regards to the former,

HIV-1 infected CD4+ T lymphocytes and

monocyte-macrophages were readily seen in both the follicular and

parafollicular areas, respectively (see Additional file 1A for

high power illustration) In contrast, only few numbers of

infected macrophages were observed in the brain as dual

labeled CD163 and HIV-1p24 cells These were identified

in the meninges and perivascular spaces, as was reported

previously [5,21] (Additional file 1B and C) This served to

highlight the lack of association between viral levels and neuropathology and differences in numbers of infected cells seen in the periphery (spleen) as compared to the brain Indeed, large numbers of infected CD4+ T lympho-cytes and monocyte-macrophages were identified in the spleen

Behavioral deficits

The behavioral phenotypes of the HIV-1 infected human-ized CD34-NSG mice were compared to uninfected

Figure 1 Timings for data acquisition in combined blood, neuroimaging, and histology assessments.

Figure 2 VL and immune profiles in HIV-1 infected humanized mice (A) Flow cytometric analysis of CD4 + and CD8 + T cells in blood of HIV-1 infected mice (n = 10) The x-axis is the weeks following HIV-1 infection (B) VL dynamics in plasma of replicate blood samples from

A Mice were bled once every 4 weeks starting from 2 nd week post-infection Mean ± SEM are shown in both A and B (C) Total HIV-1gag RNA levels in the cortex was analyzed by real time RT-PCR Data is expressed as HIV-1 RNA copies/ μg total RNA after normalizing against GAPDH (used

as an internal control) (D) For comparison, HIV-1 viral RNA in the peripheral blood at the end point is presented as viral RNA copies/ml.

controls by OFA testing [26-28] in a replicate mice Here,

mice are placed in an enclosure that permits exploration of

a new environment Spatial distribution, horizontal and

ver-tical movements are then measured to assess exploratory

behaviors By the third trial, reductions in total horizontal

distance and vertical movements in uninfected controls

reflected mouse habituation associated with loss of anxiety

and memory of the environment [27,29] Unlike uninfected controls, HIV-1 infected animals showed unremitting ac-tivity even at the third trial demonstrating continued anx-iety (Figure 3A) Implementing OFA measurements over successive trials at monthly intervals permitted inves-tigation of the level of memory with HIV-1 infection (Figure 3B) Analysis of central zone activities, expressed as

Figure 3 Effect of HIV-1 infection of humanized CD34-NSG mice on cognition (A) Schematic diagram is illustrated showing the

experimental plan used and results Three consecutive trials of OFA testing were done at 12 –13 weeks after HIV-1 infection Replicate control mice were injected with PBS for OFA testing at the same time intervals Mice were tested using 20 min sessions for three consecutive days (3 trials) Mice were bled from sub-mandibular vein under isoflurane inhalation anesthesia for flow cytometry and VL measurements Total distance travelled measured in the floor plane and vertical entries were measured in the vertical plane These reflect the exploratory and habituation behavior of the mice

in a new environment Both measurements were reduced by the 2 nd and 3 rd trials in uninfected controls reflecting habituation HIV-1 infected animals exhibit continued anxiety *, p < 0.05 compared to 1 st trial and #, p < 0.05 compared to 2 nd trial Values are mean ± SEM (B) OFA testing was performed longitudinally before infection and 4 and 8 weeks after HIV-1 infection (n = 8 for both control and infected animals) At each time point mice were tested for OFA using 20 min session for three consecutive days (3 trials) Time spent and distance traveled in the center compared to the periphery was automatically measured The ratios were analyzed to assess anxiety behavior and long term memory of environment By 8 weeks, control mice showed memory of the environment and reduced anxiety by spending more time in the central bright zone compared to HIV-1 infected mice.

* = p < 0.05 compared to trial 1 of pre-infection OFA, and # = p < 0.05 compared to the corresponding trial performed at the same time from controls Values are mean ± SEM (B, bottom right) First five minute travel paths with distance travelled for representative control and HIV-1 infected mice from trial 3 of an 8 week time point are shown.

the ratio between times spent in the margin to total time

(Figure 3B) indicated significant differences between animal

groups at 4 and 8 weeks after repeated testing and

infec-tion Control animals demonstrated memory of the

envir-onment that is not seen in the infected animal groups

Measures of central/total time 4 and 8 weeks demonstrated

that loss of anxiety readily occurred in uninfected mice but

not HIV-1 infected animals These animals showed

contin-ued anxiety by thigmotaxis [30] These tests best assess

ex-ploratory behavior, locomotor activity and anxiety-like

behavior and were employed based on the fragile nature of

the animals that would not tolerate the more rigorous

Morris water maze testing

Multispectral fluorescence imaging of immunohistology

Neuronal and glial antigens were analyzed by

multispec-tral fluorescence imaging of replicate brain regions at

study termination Images of each fluorescent spectral

component were analyzed following staining Neuronal

density was assessed using neuronal microtubule

associ-ated protein-2 (MAP2) and neurofilament (NF) Synaptic

density was assessed using synaptophysin (SYN)

Activa-tion of astrocytes was assessed using glial fibrillary acidic

protein (GFAP) Microglial activation was measured using ionized calcium binding adaptor molecule 1 (Iba-1) Oligodendrocyte numbers and integrity were measured

by myelin associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG) antigens (Figure 4) These antigens are expressed as fluorescence intensity per

μm2

(Figure 4B) Data from 20 infected animals were com-pared to 10 uninfected controls

SYN expression showed punctate and diffuse distribu-tion in cortical areas of control animals, but in case of HIV-1 infected animals, SYN labeled regions were ir-regularly shaped with significantly decreased expression and fluorescence intensity in the M2 region of the cortex (Additional file 2) with a trend towards decreased SYN

in the whisker barrels (Figure 4B) No significant altered expressions of SYN were seen in the 4 subregions of hippocampus GFAP expression was increased in 3 re-gions of hippocampus (CA1, CA3 (Additional file 2) and

DG (Figure 4)) This reflects astrocyte activation re-sponses that commonly follow HIV-1 infection Micro-glial expression, measured by Iba-1 staining, increased in the M2 region of the cortex (Additional file 2), the whis-ker barrels, and the dentate gyrus (Figure 4) In contrast,

Figure 4 Immunofluorescence staining of neuronal and glial antigens following HIV-1 infection Paraffin embedded 5 μM brain sections were immunostained with MAP2, SYN, NF, GFAP, MAG and Iba1 antibodies (A) Representative images captured at 40× magnification from whisker barrel (WB), corpus collosum (CC) and Dentate gyrus, (DG) are shown for those markers significantly deregulated after HIV-1 infection (B) Density of antigen expression quantified in different brain regions are shown for both HIV-infected and uninfected mice by multispectral imaging Decreased expression of MAG and NF in WB, CC and DG regions of infected animals compared to uninfected controls Significant increase in reactive microglia and astroglyosis (GFAP) was observed in DG Oligodendrocyte associated myelin (MOG) was reduced both in

WB and CC Genu of corpus callosum from infected animals show decreased expression of MAG, MOG and NF in infected mice Values are mean ± SEM and *denotes p < 0.05.

significant reductions in NF-positive fibers were

ob-served in the whisker barrels and corpus callosum of

HIV-1 infected mice (Figure 4) while changes in NF

ex-pression between infected and uninfected brains were

not seen in the hippocampus (Additional file 2) These

results, taken together, demonstrate that HIV-1 infected

humanized mice are distinct from previous injection

models of HIV-1 encephalitis where glial inflammatory

re-sponses drive more significant neurodegeneration [5,23]

Nonetheless, the reduced presynaptic and neurofilament

expression seen in the cortical and white matter regions of

the humanized virus-infected mice indicate ongoing

neu-rodegeneration potentially compatible with an orthograde

axonopathy

We next analyzed MOG and MAG in the white and

grey matter regions to assess oligodendrocyte and

mye-lin density MOG, is a myemye-lin component of the CNS

and responsible for maintenance of the myelin sheath,

cell adhesion and oligodendrocyte microtubule stability

MOG is localized on the oligodendrocyte cell surface

and on the outermost lamellae of mature myelin MAG

is a nervous system cell surface adhesion protein that is

involved in linking myelinated cells to neuronal axons

Like myelin, MAG inhibits axonal outgrowth and

con-tributes to the inhibitory properties of myelin We

ob-served significantly decreased MAG expression in the

whisker barrels, corpus callosum, and hippocampus

re-gions DG (Figure 4), CA1, and CA3 (Additional file 2)

in HIV infected animals Decreased MOG expression

was observed in the whisker barrels and corpus callosum

of infected animals (Figure 4) suggesting loss of axonal

fiber elements responsible for HIV-1 associated grey and

white matter damage

Correlations between antigens and viral load (VL)

Mean results provide a picture of damage focused in the

cortex and white matter tracts Hippocampus is affected

primarily with inflammation The degree of damage is

as-sociated, in measure, with the mean virus concentration in

the blood over the course of infection We hypothesized

that variability between individual mice infection level (VL

range 5 × 104to 6 × 106copies/ml blood) would lead to

differing severities of neuronal disease as reflected by

quantitative histological measures Surprisingly, there were

no significant correlations between the virus levels and the

quantitative immunohistology Only trends between MAG

reductions in the corpus callosum (r = -0.386, p = 0.1) and

GFAP in the dentate gyrus (r = -0.399, p = 0.098) were

found

Neuroimaging

Within a subset of the animals studied we acquired 1H

MRS and DTI at four week intervals from preinfection, to

16 weeks post infection in both infected animals (n = 8)

and uninfected humanized NSG mice as controls (n = 7)

In addition to comparing the groups over time to deter-mine the kinetics of brain pathology in this model, results for the individual infected animals were tested for correla-tions to virus levels and histopathology to aid in the inter-pretation of changes observed

1

H MRS

Mean metabolic consequences of HIV-1 infection as com-pared to uninfected animals can be seen in the cerebral cortex (Figure 5A, B) Reduced NAA and increased GABA were seen at 12 weeks and reversed at 16 weeks in the cerebral cortex but not in the cerebellum (Figure 5C, D) Notably, a large increase in lactate was observed in the cerebral cortex at 16 weeks after infection (Figure 5A, B) Other metabolic effects of infection include reductions of creatine and choline in the infected cerebral cortex over time (Additional file 3) which is not seen in the cerebel-lum (Additional file 4) These changes are subtle and not significantly different from what is generally observed in uninfected animals Indeed, in the cerebral cortex of in-fected animals the reductions of choline and creatine were significantly different only from the preinfection levels of the same animals

Correlations between cortical metabolites and VL

To further examine the relationship between blood virus levels and brain pathology, we investigated the correla-tions between end-stage (16 week post infection) metab-olite concentrations and the mean blood virus level over the course of infection Only one of the metabolites had

a significant correlation with mean virus concentration, lactate (r = 0.857, p = 0.024) with a trend towards correl-ation with myoinostitol (r = -0.714, p = 0.088) While in-creasing lactate would be expected with higher viral loads, decreasing myoinostitol with higher viral loads is unexpected, as increased neuroinflammation is pre-sumed to be associated with increased myoinostitol In order to further explore these effects, we investigated the correlations between 16 week post infection metab-olite concentrations and quantitative immunohistology

in the cerebral cortex

1

H MRS cortical metabolites and neuronal, oligodendrocyte and glial markers

The correlations between metabolite concentrations at the end-point of the study with cortical markers for synap-tic density (SYN), glial activation (Iba-1, GFAP), and oligodendrocyte-associated proteins density (MAG, MOG) were measured between the cortex spectrum and the quantitative histology from the M2 region of the cortex Significant correlations were found between reduced MAG in the M2 region of the cerebral cortex (M2c) and reduced 16 week concentrations of the metabolites

Figure 5 Metabolite levels (Means ± SEM) expressed as a percentage of total signal acquired over time from 1 H MRS scans of (red) HIV-1 infected (n = 8) and (black) uninfected humanized mouse controls (n = 7) (A) Region selected for spectral acquisition from the cerebral cortex (B) Selected metabolite levels are measured from the cerebral cortex Time zero, in infected mice is before infection with

subsequent spectra acquired every four weeks to 16 weeks in both infected and control mice (C) Region selected for acquisition of spectrum from cerebellum (D) The same metabolites as shown in (B) Time courses of all metabolites are shown in Additional files 3 and 4 *p < 0.05 control versus HIV-1 infected mice, +p < 0.05 vs time zero in control mice, (red “+” symbol) p < 0.05 versus preinfection in infected mice.

glutamate (r = 0.929, p = 0.024), myoinostitol (r = 0.857,

p = 0.024) as well as a trend for increased taurine (r =

-0.750, p = 0.066) (Figure 6I-K) In addition, there was a

trend for increased NAA (r = -0.714, p = 0.088) and creatine

(r = -0.750, p = 0.066) to correlate with reduced M2c MAG

(Figure 6A, E, I-L) Similar, but opposite relationships

existed between SYN and NAA (r = 0.893, p = 0.012), and

creatine (r = 0.857, p = 0.024) (Figure 6B, F) In addition,

there was a tendency for increased GFAP levels to correlate

with reduced NAA (r = -0.714, p = 0.088) (Figure 6D)

Fi-nally, it was also found that levels of lactic acid showed a

trend towards correlating with MOG (r = 0.750, p = 0.066)

(Figure 6H) This provides an overall picture of cortical

me-tabolism changes from HIV-1 infection showing that

pro-cesses leading to synaptic loss are associated with loss of

NAA and creatine, neuroinflammation being associated with NAA loss, while reduced MAG are associated with increases in NAA, creatine, taurine, and reductions in glutamate and myoinostitol Certainly, these associations cannot be over interpreted, but are generally in agreement with expected metabolic effects of inflammation, synaptic loss, and loss of oligodendrocytes-associated proteins in the cortex of these infected animals

DTI

DTI metrics investigated include FA, Dav, longitudinal dif-fusivity (λl) and transverse diffusivity (λt) (see Materials and Methods) in brain regions These were measured in the hippocampus [CA1, CA2, CA3 and dentate gyrus (DG)], in cortex (whisker barrels, middle cerebral cortex,

Figure 6 Correlations between cortical metabolites at 16 weeks after infection and quantitative multispectral histology The latter was measured in the M2 region of the cerebral cortex (A-D) NAA versus MAG, SYN, MAP2 and GFAP respectively are shown (E-G) Creatine versus MAG, SYN, and MAP2 are illustrated (H) Lactate versus MOG (I-L) MAG versus myoinostitol, taurine, glutamate, and glutamate and glutamine, respectively were analyzed in these data sets.

Figure 7 (See legend on next page.)

M2 cortical region, frontal cortex), and white matter regions

(splenium and genu of the corpus callosum) (Figure 7A)

Metrics of uninfected control and HIV-1 infected animals

were compared over time Results of individual mice and

brain regions were evaluated independently with

correc-tion for multiple comparisons The most consistent and

notable changes were seen as reduced FA in hippocampus

and cortical regions relative to the uninfected controls

(Figure 7B) Many of these differences are accompanied by

increase in FA in the uninfected humanized mice that did

not manifest in the infected mice It is also notable that

the cause(s) of such a reduction in FA in some regions are

reversible, including the dentate gyrus and splenium of

the corpus callosum, possibly indicating transient

in-flammatory processes occurring in these regions during

the course of infection Other notable alterations included

increased diffusivity in white matter (Additional files 5, 6

and 7)

DTI and VL

End-point values of DTI metrics were tested for

correl-ation with mean viral loads in the individual infected

mice (Figure 8) Correlations with virus level were weak

and found only in cortical regions (M2 region of the

cor-tex and whisker barrels) in multiple DTI measures, but

only a trend of a single correspondence in DG and no

other regions This suggests that the cortical regions are

the primary site of neuronal damage due to

multifactor-ial HIV-1 induced injuries Exactly which factors are

pri-mary causes of this damage will be a matter for further

exploration Nevertheless, further details regarding

spe-cific histological abnormalities and DTI metrics were

then explored in the cortical regions, hippocampus, and

corpus callosum

DTI and neuronal, oligodendrocyte and glial markers

End-point values of DTI metrics were correlated with

quantitative histopathology in the individual infected

mice (Figure 9) Correlations were considered between

the DTI metrics within spatial subregions used for

histo-logical analysis which included cortical regions (M2

region of the cortex (M2c), whisker barrel regions (WB)), hippocampus regions (CA1, CA2, CA3 and the dentate gyrus (DG)), genu of the corpus callosum, cerebellum, and pons-medulla (brainstem) as delineated in Figure 7 The most significant correlations were found in the cortical re-gions, similar to virus level It was observed that in the whisker barrel region increasing mean diffusivity and de-creasing FA correlate with inde-creasing MAG and with in-creasing Iba-1 In addition, FA reduced with NF loss The CA2 region of the hippocampus shows correlation of in-creased diffusivity, especially the longitudinal diffusivity with decreasing SYN with a similar trend with MAP2 No significant correlations were found in the corpus callosum, brainstem, or the cerebellum

Discussion

There are expected similarities and noted differences be-tween the neurological consequences of HIV-1 infection

in humans and in this mouse model As for the former, chronic infection in the mice leads to viral invasion of the central nervous system and ingress of monocyte-derived macrophages first into the meninges then the perivascular spaces Behavioral abnormalities follow but more subtle in mice They are coincident with CD4+ T cell loss and high peripheral viral loads Notably, mouse infection leads to micro- and astro- gliosis and neuronal loss that, like in humans, are region and animal specific Indeed, levels of infection and histopathological findings

in brain vary Such variability rests in the extent of in-flammation and neuronal damage We have not yet ob-served a true multinucleated giant cell encephalitis in mice as is often present in humans and likely due to that primary microglia and astrocytes are rodent-in-origin and as such cannot be infected with the virus Nonethe-less, the finding in these rodents underlies the central tenet of human disease in that few infected cells in the brain can give rise to more wide spread alterations in neural function underpinning the role of a virus-induced metabolic encephalopthy Importantly, it is the engage-ment of the innate and adaptive immune system trig-gered by the virus that are not only operative in the

(See figure on previous page.)

Figure 7 DTI metrics (A) Brain regions analyzed for DTI metrics (B) Fractional anisotropy (mean ± SEM) in (top) CA1, CA3, and dentate gyrus (from left to right) and (bottom) frontal cortex, middle cerebral cortex, and splenium of the corpus callosum (from left to right) as shown in (A) (red “*” symbol) p < 0.05 control (n = 7) vs infected (n = 8) mice, +

p < 0.05 versus time zero in control mice, (red “+” symbol) p < 0.05 versus pre-infection in HIV-1 infected animals.

Figure 8 Correlations between cortical DTI metrics and mean plasma VL 16 weeks after HIV-1 infection.